The Oxford Regional Prospective Study

Abstract

OBJECTIVE—To describe longitudinal variations in pubertal hormonal variables in subjects with and without microalbuminuria (MA).

RESEARCH DESIGN AND METHODS—Blood samples collected annually from subjects recruited at diagnosis of type 1 diabetes and followed prospectively through puberty (median follow-up 9.3 years, range 4.7–12.8) were analyzed for total and free IGF-I, IGF binding protein-1, testosterone, sex hormone-binding globulin, and HbA1c. A total of 55 subjects who developed MA (MA+ group) were compared with 55 age-, sex-, and duration-matched control subjects who did not develop MA (MA− group).

CONCLUSIONS—Poor glycemic control and differences in IGF-I levels and androgens, particularly in female subjects, accompany development of MA at puberty. These differences may in part account for the sexual dimorphism in MA risk during puberty and could relate to disease progression.

Microalbuminuria (MA) is a marker of incipient nephropathy in adult subjects with type 1 diabetes (1). There is an unequivocal relationship between poor glycemic control and the development of MA (2). However, independent of poor glycemic control, early diabetic complications risk is increased with the onset of puberty (3). In addition, MA risk is twofold greater in pubertal female patients compared with male patients (3), in contrast to lifetime risk of diabetic nephropathy, which is greater in male patients (4). Sexual dimorphism is also present during puberty for risk of attenuated growth, weight gain, and retinopathy, and again these observations are independent of glycemic control (5–7). These data suggest that the development of diabetic microvascular complications may be associated with abnormalities in hormonal variables related to pubertal development.

Cross-sectional data describe abnormalities of the growth hormone (GH)/ IGF-I axis in relation to the development of diabetic complications (8). In type 1 diabetes, bioavailability of circulating IGF-I is low and GH secretion exaggerated (9,10), and these abnormalities may be more marked in female patients (10). Despite evidence of hepatic GH resistance, integrity of GH pathways in other tissues remain intact, and both elevated GH and local paracrine IGF-I generation have been implicated in the development of diabetic nephropathy in humans and rats (11,12). Hyperandrogenism and low sex hormone-binding globulin (SHBG) levels in female patients have also been linked to MA risk (13), and this may relate to the reported increased prevalence of polycystic ovarian syndrome (PCOS) in young women with type 1 diabetes (5).

Cross-sectional studies during puberty are easily confounded by variables such as diabetes duration, pubertal stage, and inadequate selection of control subjects. A further confounding factor is the variability in urine albumin excretion, since it is thought that MA may resolve in up to 50% of cases during adolescence (3). To address these issues, we report longitudinal changes in IGF-I and sex steroids in a cohort of well-characterized male and female subjects who were recruited at diagnosis of type 1 diabetes and followed longitudinally through puberty, comparing those who went on to develop MA against carefully matched control subjects without MA.

RESEARCH DESIGN AND METHODS

Study design

Subjects.

The Oxford Regional Prospective Study of Childhood Diabetes (ORPS) was established in 1986, and the characteristics of the cohort have been previously described (3). Children under 16 years of age were recruited within 3 months of diagnosis of type 1 diabetes and were assessed at the end of the first year from diagnosis and annually thereafter. Assessments consisted of measurements of height, weight, and three consecutive early-morning (first void) urine specimens for the albumin-to-creatinine ratio (ACR). Blood samples were collected for the central measurement of HbA1c, and annual nonfasting blood samples were stored on each patient. Ethical approval was obtained from the regional ethics committees, with written consent from the parents and assent from the children.

MA was defined as an ACR >3.5 mg/mmol in male subjects and >4.0 mg/mmol in female subjects and <35 mg/mmol in two of three consecutive early-morning urine collections (3). This corresponded to an albumin excretion rate of between 20 and 200 mcg/min, as determined in 304 timed overnight urine samples using linear regression equations.

Of the 494 subjects recruited, 63 had an ACR that fell within our definition of MA on at least one annual assessment. A total of 55 subjects had sufficient blood samples for analysis, and these were designated as case subjects (MA+ group). Case subjects were matched for age, sex, and duration of diabetes to control subjects selected from the remaining subjects who, to date, have no evidence of MA (MA− group). For each subject, all available longitudinally collected blood samples were analyzed for the relevant hormones.

Methods

Auxology.

BMI standard deviation scores (SDSs) were calculated using data based on the British 1990 Growth Reference and Cole’s LMS method (14).

Albumin assay.

Details of sample collection and storage were reported previously (4). Albumin was measured centrally by an enzyme-linked immunosorbent assay method (3). The within- and between-assay coefficients of variation (CVs) was 6 and 12%, respectively.

Creatinine.

HbA1c.

HbA1c was measured centrally, initially by an electrophoretic method (Ciba Corning Diagnostics, Halstead, U.K.), which was replaced by high-performance liquid chromatography (DIAMAT; Bio-Rad, U.K.) in 1992 (15). The relationship between the two methods has been described previously (3). The within-batch CV was 2.2 and 1.3% at a level of 9.8 and 10.1%, respectively. The between-batch CV was 3.5 and 2.2% at 5.6 and 10.1%, respectively. The normal range for the assay is 4.4–6.2%.

Total IGF-I.

Total IGF-I levels were determined by radioimmunoassay (RIA) after acid-acetone extraction using rabbit anti-serum developed by L. Underwood (North Carolina University, Chapel Hill, NC). The assay was standardized against a pool of normal human serum, defined as containing 1.0 units IGF-I/ml, equivalent to 159 ng/ml of a purified preparation of IGF-I (17). The intra- and interassay CVs were 6.2 and 3.5%, respectively.

Statistical methods

All data were normally distributed, except IGFBP-1, testosterone, SHBG, and free androgen index (FAI; FAI = testosterone × 100/SHBG), which were log transformed to allow parametric analyses. Levels of each hormone were compared between groups using an ANOVA model. To adequately consider the effects of puberty, a Cox proportional hazards model was fitted with duration of diabetes as the time variable, MA as the outcome, and pubertal onset as a time-dependent covariate (using age 11 years as a surrogate marker for puberty onset). For this model, data from each subject was summarized by calculating the mean of all measurements. The covariates examined included HbA1c, free IGF-I, daily insulin dose, and testosterone SDS. Testosterone SDSs were used to allow both sexes to be considered together and were derived from comparison of data against comparable age- and sex-matched nondiabetic subjects using the Growth Analyser Program (Dutch Growth Foundation). To display the longitudinal changes in hormone levels over time, we used a multilevel modeling software (MLwiN version 1.0 beta; Institute of Education, London) (19). This is an extension of multiple regression, using repeated-measures data and analyses within and between individual effects, allowing consideration of individual curves and their summation by predefined groups. SPSS version 10.0 was used for analysis. Data are presented as the mean ± SD or median (interquartile range). A P value <0.05 was considered significant.

RESULTS

Cohort characteristics

There were no significant differences between the groups in demographic characteristics, BMI SDS, and insulin daily dose (Table 1). Of the 55 case subjects, 33 (60%) had transient MA (defined as MA present for 1 year with normoalbuminuria the following year) and 22 (40%) developed persistent MA (defined as MA present for ≥2 consecutive years).

Free IGF-I.

Free IGF-I levels were lower in the MA+ compared with the MA− group (1,678 ± 745 vs. 1,887 ± 906 ng/l, P = 0.004). In female subjects, the difference between the groups was highly significant (1,767 ± 634 vs. 2010 ± 881 ng/ l, P = 0.002) (Fig. 1A and B), but this was not the case in males (1,552 ± 946 vs. 1,668 ± 930 ng/l, P = 0.77). In females with similar HbA1c levels, free IGF-I levels were consistently lower in those with MA compared with those without (Fig. 1C).

IGFBP-I.

No significant differences in IGFBP-I levels were found

Testosterone, SHBG, and FAI.

In female subjects, testosterone levels (median [interquartile range]) were higher in the MA+ compared with the MA− group (1.3 [0.9–1.6] vs. 1.1 [0.7–1.3] mmol/l, P = 0.04), as was the FAI (2.4 [1.3–4.0] vs. 2.0 [1.0–3.2], P = 0.03) (Fig. 1D and E). At similar HbA1c levels, FAI was consistently higher in those with MA compared with those without MA (Fig. 1F). In male subjects, no differences were found. No relationship was seen with daily insulin dose and SHBG levels for either sex (nonfasting samples).

Proportional contribution of hormonal covariates and HbA1c to probability of developing MA

Independent of puberty, the probability of having MA was associated with lower free IGF-I levels (P = 0.01, Exp[B] = 0.999, 95% CI 0.998–1.0; i.e., a 10% increase in probability with a reduction in free IGF-I levels by 100 ng/l), with higher testosterone SDS (P < 0.001, Exp[B] = 3.9, 95% CI 2.1–7.3; i.e., by a factor of 3.9 for a unit rise), and with poor glycemic control (P < 0.001, Exp[B] = 3.5, 95% CI 2.1–5.8; i.e., by a factor of 3.5 for a 1% increase in mean HbA1c levels). When the sexes were considered separately, the findings for female subjects were similar to when both sexes were considered together, but for male subjects, the probability of having MA was associated with poor glycemic control only (P = 0.04).

CONCLUSIONS

There is little longitudinal data on the natural history of MA in children with type 1 diabetes. Data from ORPS has shown that the cumulative probability of developing MA after 9 years’ diabetes duration is 40% (3). Follow-up of the cohort shows that MA persists in 40% of these subjects, with persistence being unequivocally related to poor glycemic control. However, the difference in HbA1c between those with transient MA and control subjects was not significant, and the increasing prevalence of MA during puberty cannot be entirely explained by HbA1c. Puberty and sex are also independent risk factors for the development of MA (3). Pubertal onset is associated with an acceleration in the urine albumin excretion rate compared with the years during prepuberty, and this may be predictive of MA (20), and female subjects are at a twofold greater risk of MA compared with male subjects (3). This marked sexual dimorphism is also apparent in other areas at puberty, as evidenced by the increased risk in adolescent female subjects for developing retinopathy (7), obesity (5), and attenuated growth (6). These data suggest hormonal changes during puberty may effect the development of MA in susceptible individuals.

In female subjects and, to a lesser extent in male subjects, those with MA had lower total and free IGF-I levels than their control subjects, and this could by implication relate to variation in GH levels. In type 1 diabetes, relative portal insulinopenia (caused by failure to administer insulin directly into the portal vein) results in impaired hepatic generation of total and free IGF-I (9), leading to a lack of negative feedback drive for GH hypersecretion, over and above that seen in normal puberty (10). Thus, circulating GH levels are increased while circulating IGF-I levels remain low, and these changes may be more apparent in female than in male subjects (10). The integrity of GH pathways in tissues other than the liver are thought to remain intact, and both GH hypersecretion and local paracrine IGF-I production have been implicated in the pathophysiology of diabetic nephropathy. In animal models, GH increases renal blood flow (12), renal expression of IGF-I, and renal size (21), with IGF-I expression within the kidney correlating directly with diabetic nephropathy-like changes (22). Selective GH blockade leads to reduced renal expression of IGF-I, renal size, GFR, and urine albumin excretion (22). In humans, GH hypersecretion and local paracrine IGF-I generation have been associated with the development of diabetic nephropathy (11) and retinopathy (23,24) in cross-sectional studies. Acromegaly is associated with increases in GFR and urinary albumin excretion (25), whereas specific GH blockade leads to normalization of these factors (25). However, to date no renal physiology data exist on such intervention in type 1 diabetic subjects.

Our findings of raised androgens in those with MA, particularly in female subjects, confirms findings in previous cross-sectional studies (13). It is probable, but unproven, that the elevated androgens in female subjects derive from the ovary, and that ovarian hyperandrogenism is a principal feature of PCOS. The relationship between ovarian hyperandrogenism and polycystic morphologic changes of the ovary is much debated, but in one study of adolescent girls with type 1 diabetes, up to 50% had ovarian changes characteristic of PCOS, in contrast to 20–30% in the general population (5).

Ovarian hyperandrogenism in PCOS is related to insulin resistance and peripheral hyperinsulinemia (26), although elevated GH and reduced IGF-I levels could also theoretically affect ovarian function (27). The GH-associated increase in insulin resistance during puberty in type 1 diabetes may be the principal cause of the ovarian hyperandrogenism. However, other genetic variation in factors leading to insulin resistance may also be important because insulin resistance can predate the appearance of MA (28), with familial clustering of diabetic nephropathy occurring in association with changes in insulin resistance (29).

We currently have no direct measures of insulin resistance in our case-control series. Insulin doses were similar in case and control subjects, yet HbA1c was higher in case subjects. This might indicate underinsulinization rather than insulin resistance, and poor control might lead to abnormalities of both the GH-IGF-I axis and MA. However, free IGF-I levels were lower and FAI higher in those with MA, independent of HbA1c (Fig. 1C and F), suggesting that these hormone changes and any associated increase in GH levels may influence the expression of MA in genetically susceptible individuals as they move through puberty. Thus, background variability in common genetic polymorphisms, such as in the IGF-I gene and the androgen receptor gene, may contribute to MA risk; however, this has yet to be determined. The role of changes in IGF-I levels and FAI in the pathogenesis of MA cannot be further elucidated by these studies. However, the differences in the degree of abnormality seen in those with persistent and transient MA may be predicted by lower HbA1c (20) and less profound abnormalities of the GH-IGF-I and ovarian axes.

In summary, the development of MA at puberty may reflect not only poor glycemic control but also changes in the GH-IGF-I axis and ovarian function. Changes in pubertal hormonal variables differ in those with MA, particularly in female patients, and these differences may relate to disease progression. Aggressive insulin therapy has been shown to reduce diabetic complication risk (2). However, this may confer increased weight gain and hypoglycemia (2) and predispose to the detrimental effects of peripheral hyperinsulinemia, such as the development of ovarian hyperandrogenism. Increasing the insulin dose to overcome insulin resistance may lead to further weight gain (2) and, in female patients, a cycle of insulin omission to lose weight (30). This pattern of insulin misuse has been linked to the increased risk of diabetic complications in adolescent girls during puberty (31). Our observations suggest alternative therapy directed at underlying mechanisms, such as insulin sensitizers, IGF-I therapy, and anti-androgen therapy, might also be explored in attempts to reduce progression of microvascular complications in high-risk individuals during puberty.

Free IGF-I in female case subjects (MA+) and control subjects (MA−) across years relative to appearance of MS, time 0 denoting first appearance of MA (A), across age (B), and across corresponding HbA1c values (C). FAI levels in case subjects (MA+) and control subjects (MA−) are also shown across years relative to appearance of MA, time 0 denoting first appearance of MA (D), across age (E), and across corresponding HbA1c values (F) (derived from multilevel modeling, dotted lines representing ± SE).

Urine ACR in case subjects with persistent and transient MA and their matched normoalbuminuric control subjects (MA−) across years relative to the onset of MA, time 0 denoting first appearance of MA. ACR is expressed as the log of the geometric mean of three consecutive early-morning first-void samples (derived from multilevel modeling, dotted lines representing the mean ± SE).

Summary of hormonal variables for those with transient MA and persistent MA and their matched normoalbuminuric control subjects (MA−)

Acknowledgments

This study was supported by the Novo Nordisk Center for Research in Growth and Regeneration, Aarhus University, Denmark (Danish Health Research Council Grant 9700592); the Institute of Experimental Clinical Research, University of Aarhus, Denmark; and the Hørslev Foundation. R.A. is a Novo Nordisk U.K. Research Foundation fellow. The ORPS is funded by the British Diabetic Association.

We acknowledge the Juvenile Diabetes Research Foundation; the study field workers; the laboratory assistance of Angie Watts and Dot Harris; the Barts-Oxford Study field workers; and the pediatricians, physicians, and diabetes nurse specialists in the Oxford region. In addition, We are indebted to Joan Hansen for skilled technical assistance.

References

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